Children less than 2 years of age do not mount an immune response to most polysaccharide vaccines, so it has been necessary to render the polysaccharides immunogenic by chemical conjugation to a protein carrier. Coupling the polysaccharide, a T-independent antigen, to a protein, a T-dependent antigen, confers upon the polysaccharide the properties of T dependency including isotype switching, affinity maturation, and memory induction.
Streptococcus pneumoniae is a Gram-positive bacterium responsible for considerable morbidity and mortality (particularly in the young and aged), causing invasive diseases such as pneumonia, bacteraemia and meningitis, and diseases associated with colonisation, such as acute Otitis media. The rate of pneumococcal pneumonia in the US for persons over 60 years of age is estimated to be 3 to 8 per 100,000. In 20% of cases this leads to bacteraemia, and other manifestations such as meningitis, with a mortality rate close to 30% even with antibiotic treatment.
Pneumococcus is encapsulated with a chemically linked polysaccharide which confers serotype specificity. There are 90 known serotypes of pneumococci, and the capsule is the principle virulence determinant for pneumococci, as the capsule not only protects the inner surface of the bacteria from complement, but is itself poorly immunogenic. Polysaccharides are T-independent antigens, and cannot be processed or presented on MHC molecules to interact with T-cells. They can however, stimulate the immune system through an alternate mechanism which involves cross-linking of surface receptors on B cells.
It was shown in several experiments that protection against invasive pneumococci disease is correlated most strongly with antibody specific for the capsule, and the protection is serotype specific.
Streptococcus pneumoniae is the most common cause of invasive bacterial disease and Otitis media in infants and young children. Likewise, the elderly mount poor responses to pneumococcal vaccines [Roghmann et al., (1987), J. Gerontol. 42:265-270], hence the increased incidence of bacterial pneumonia in this population [Verghese and Berk, (1983) Medicine (Baltimore) 62:271-285].
Conjugation of Streptococcus pneumoniae saccharides using cyanoborohydride ions is known, for example WO87/06838. However cyanoborohydride ions have several disadvantages including a relatively slow reaction time and the possible contamination of the product with cyanide (Ahmed F. et al J. Org. Chem., 1996, 61:3849-3862). The slow reaction time using this reagent was addressed in EP1035137 which describes an attempt to improve the reaction time of this conjugation process through use of microwave radiation.
Ahmed et al have described the reductive amination of aldehydes and ketones with sodium triacetoxyborohydride (NaBH(OAc)3) (J. Org. Chem., 1996, 61:3849-3862). Similarly the reductive amination of carbohydrates using NaBH(OAc)3 was described by Dalpathado et al (Anal. Bioanal. Chem (2005) 281:130-1137).
Accordingly the present invention provides an improved process for conjugation of an antigen by reductive amination using triacetoxyborohydride (BH(OA)3) anions at the reducing agent. The inventors have discovered that this reducing agent is suitable for use to conjugate bacterial saccharides to carrier proteins; in particular this is quicker than the equivalent reaction using cyanoborohydride ions and does not produce toxic by-products.